Novel recombinant and mutant adenoviruses

ABSTRACT

The present invention provides novel viral vectors. In one embodiment, the present invention provides mutant and recombinant bovine adenoviruses having a deletion and/or insertion of DNA in the early gene region 4 (E4). In another embodiment, the present invention provides mutant and recombinant bovine adenovirus 1 viruses having a deletion and/or insertion of DNA in the early gene region 3 (E3). The present invention also contemplates the use of the viral vectors for vaccination, gene therapy or other applications as suitable.

The benefit of the Apr. 9, 1999 filing date of Provisional ApplicationNo. 60/128,766 is claimed.

FIELD OF THE INVENTION

The present invention relates to viral vectors for vaccination ofanimals. In particular, the present invention pertains to viral vectorshaving insertion sites for the introduction of foreign DNA.

BACKGROUND OF THE INVENTION

The adenoviruses cause enteric or respiratory infection in humans aswell as in domestic and laboratory animals.

Inserting genes into adenoviruses has been accomplished. In the humanadenovirus (HuAd) genome there are two important regions: E1 and E3 inwhich foreign genes can be inserted to generate recombinantadenoviruses.

This application of genetic engineering has resulted in several attemptsto prepare adenovirus expression systems for obtaining vaccines.Examples of such research include the disclosure of U.S. Pat. No.4,510,245 of an adenovirus major late promoter for expression in a yeasthost; U.S. Pat. No. 4,920,209 of a live recombinant adenovirus type 7with a gene coding for hepatitis-B surface antigen; European patent No.389,286 of a non-defective human adenovirus 5 recombinant expressionsystem in human cells; and published International application No. WO91/11525 of live non-pathogenic immunogenic viable canine adenovirus ina cell.

However, because they are more suitable for entering a host cell, anindigenous adenovirus vector would be better suited for use as a liverecombinant virus vaccine in different animal species compared to anadenovirus of human origin. For example, bovine adenovirus-basedexpression vectors have been reported for bovine adenovirus 3 (BAV-3)(see U.S. Pat. No. 5,820,868).

Bovine adenoviruses (BAVs) comprise at least nine serotypes divided intotwo subgroups. These subgroups have been characterized based onenzyme-linked immunoassays (ELISA), serologic studies withimmunofluorescence assays, virus-neutralization tests, immunoelectronmicroscopy and by their host specificity and clinical syndromes.Subgroup 1 viruses include BAV 1, 2, 3 and 9 and grow relatively well inestablished bovine cells compared to subgroup 2 viruses which includeBAV 4, 5, 6, 7 and 8.

BAV-3 was first isolated in 1965 and is the best characterized of theBAV genotypes and contains a genome of approximately 35 kilobases. Thelocations of hexon and proteinase genes in the BAV-3 genome have beenidentified and sequenced.

Genes of the bovine adenovirus 1 (BAV-1) genome have also beenidentified and sequenced. However, the location and sequences of othergenes such as certain early gene regions in the BAV genome have not beenreported.

The continued identification of suitable viruses and gene insertionsites are valuable for the development of new vaccines. The selection of(i) a suitable virus and (ii) the particular portion of the genome touse as an insertion site for creating a vector for foreign geneexpression, however, pose a significant challenge. In particular, theinsertion site must be non-essential for the viable replication of thevirus, as well as its operation in tissue culture and in vivo. Moreover,the insertion site must be capable of accepting new genetic material,while ensuring that the virus continues to replicate.

What is needed is the identification of novel viruses and gene insertionsites for the creation of new viral vectors.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides recombinant viruses.while not limited to a particular use, these recombinant viruses can beused to generate vaccines.

While not limited to a particular virus, in one embodiment the presentinvention provides a recombinant virus comprising a foreign DNA sequenceinserted into the E4 gene region of a bovine adenovirus. In a preferredembodiment, the insertion is to a non-essential site. In anotherembodiment, the present invention provides a recombinant viruscomprising a foreign DNA sequence inserted into the E3 gene region of abovine adenovirus 1. In a preferred embodiment, the insertion is to anon-essential site.

While not limited to its ability to replicate, in a preferredembodiment, the recombinant virus is replication competent. Likewise,while not limited to the foreign DNA to be inserted, in a preferredembodiment, the foreign DNA encodes a polypeptide and is from a virus orbacteria selected from the group consisting of bovine rotavirus, bovinecoronavirus, bovine herpes virus type 1, bovine respiratory syncytialvirus, bovine para influenza virus type 3 (BPI-3), bovine diarrheavirus, bovine rhinotracheitis virus, bovine parainfluenza type 3 virus,Pasteurella haemolytica, Pasteurella multocida and/or Haemophilussomnus. In another preferred embodiment, the foreign DNA encodes acytokine. In a further preferred embodiment, the polypeptide comprisesmore than ten amino acids and is antigenic. Finally, in a particularlypreferred embodiment, the foreign DNA sequence is under the control of apromoter located upstream of the foreign DNA sequence.

The present invention also contemplates mutant viruses. While notlimited to a particular mutant virus, in one embodiment, the mutantvirus comprises a deletion of at least a portion of the E4 gene regionof a bovine adenovirus. In a preferred embodiment, the deletion is of anon-essential site. In another embodiment, the virus comprises adeletion of at least a portion of the E3 gene region of a bovineadenovirus 1. In a preferred embodiment, the mutant virus is replicationcompetent. In a further preferred embodiment, at least one open readingframe of the relevant gene region of the bovine adenovirus is completelydeleted.

In yet another embodiment, the present invention provides a method forpreparing a recombinant virus comprising inserting at least one foreigngene or gene fragment that encodes at least one antigen into the genomeof a virus wherein said gene or gene fragment has been inserted into theearly gene region 4 of a bovine adenovirus or inserted into the earlygene region 3 of bovine adenovirus 1. In a preferred embodiment, themethod includes the insertion of at least a part of the genome of avirus into a bacterial plasmid, transforming said bacteria with saidplasmid, and incubating said bacteria at approximately 25° C.

In another embodiment, the present invention provides vaccines. Whilenot limited to a particular vaccine, in one embodiment, the vaccinescomprise the recombinant viruses described above.

The present invention also contemplates methods of vaccination,including, but not limited to, the introduction of the above-describedvaccines to an animal.

Definitions

The term, “animal” refers to organisms in the animal kingdom. Thus, thisterm includes humans, as well as other organisms. Preferably, the termrefers to vertebrates. More preferably, the term refers to bovineanimals.

A “vector” is a replicon, such as a plasmid, phage, cosmid or virus, towhich another DNA sequence may be attached so as to bring about theexpression of the attached DNA sequence.

For purposes of this invention, a “host cell” is a cell used topropagate a vector and its insert. Infecting the cell can beaccomplished by methods well known to those skilled in the art, forexample, as set forth in Transfection of BAV-1 DNA below.

A DNA “coding sequence” is a DNA sequence which is transcribed andtranslated into a polypeptide in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, procaryotic sequences, cDNA fromeucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian)DNA, viral DNA, and even synthetic DNA sequences. A polyadenylationsignal and transcription termination sequence can be located 3′ to thecoding sequence.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase or an auxiliary protein and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is in close proximity to the 5′terminus by the translation start codon (ATG) of a coding sequence andextends upstream (5′ direction) to include the minimum number of basesor elements necessary to facilitate transcription at levels detectableabove background. Within the promoter sequence will be found atranscription initiation site, as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eucaryotic promoters will often, but not always, contain “TATA” boxesand “CAAT” boxes, conserved sequences found in the promoter region ofmany eucaryotic organisms.

A coding sequence is “operably linked to” or “under the control of”promoter or control sequences in a cell when RNA polymerase willinteract with the promoter sequence directly or indirectly and result intranscription of the coding sequence into mRNA, which is then translatedinto the polypeptide encoded by the coding sequence.

A “double-stranded DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itsnormal, double-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes, for example,double-stranded DNA found in linear DNA molecules (e.g., restrictionfragments of DNA from viruses, plasmids, and chromosomes), as well ascircular and concatamerized forms of DNA.

A “foreign DNA sequence” is a segment of DNA that has been or will beattached to another DNA molecule using recombinant techniques whereinthat particular DNA segment is not found in association with the otherDNA molecule in nature. The source of such foreign DNA may or may not befrom a separate organism than that into which it is placed. The foreignDNA may also be a synthetic sequence having codons different from thenative gene. Examples of recombinant techniques include, but are notlimited to, the use of restriction enzymes and ligases to splice DNA.

An “insertion site” is a restriction site in a DNA molecule into whichforeign DNA can be inserted.

For purposes of this invention, a “homology vector” is a plasmidconstructed to insert foreign DNA sequence in a specific site on thegenome of an adenovirus.

The term “open reading frame” or “ORF” is defined as a genetic codingregion for a particular gene that, when expressed, can produce acomplete and specific polypeptide chain protein.

A cell has been “transformed” with exogenous DNA when such exogenous DNAhas been introduced inside the cell membrane. Exogenous DNA may or maynot be integrated (covalently linked) to chromosomal DNA making up thegenome of the cell. In procaryotes and yeasts, for example, theexogenous DNA may be maintained on an episomal element, such as aplasmid. A stably transformed cell is one in which the exogenous DNA hasbecome integrated into the chromosome so that it is inherited bydaughter cells through chromosome replication. For mammalian cells, thisstability is demonstrated by the ability of the cell to establish celllines or clones comprised of a population of daughter cells containingthe exogenous DNA.

A “replication competent virus” is a virus whose genetic materialcontains all of the DNA or RNA sequences necessary for viral replicationas are found in a wild-type of the organism. Thus, a replicationcompetent virus does not require a second virus or a cell line to supplysomething defective in or missing from the virus in order to replicate.A “non-essential site in the adenovirus genome” means a region in theadenovirus genome, the polypeptide product or regulatory sequence ofwhich is not necessary for viral infection or replication.

Two polypeptide sequences are “substantially homologous” when at leastabout 80% (preferably at least about 90%, and most preferably at leastabout 95%) of the amino acids match over a defined length of themolecule.

Two DNA sequences are “substantially homologous” when they are identicalto or not differing in more that 40% of the nucleotides, more preferablyabout 20% of the nucleotides, and most preferably about 10% of thenucleotides.

A virus that has had a foreign DNA sequence inserted into its genome isa “recombinant virus,” while a virus that has had a portion of itsgenome removed by intentional deletion (e.g., by genetic engineering) isa “mutant virus.”

The term “polypeptide” is used in its broadest sense, i.e., any polymerof amino acids (dipeptide or greater) linked through peptide bonds.Thus, the term “polypeptide” includes proteins, oligopeptides, proteinfragments, analogs, muteins, fusion proteins, etc.

“Antigenic” refers to the ability of a molecule containing one or moreepitopes to stimulate an animal or human immune system to make a humoraland/or cellular antigens specific response. An “antigen” is an antigenicpolypeptide.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to the composition or vaccine of interest. Usually, such aresponse consists of the subject producing antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells directedspecifically to an antigen or antigens included in the composition orvaccine of interest.

The terms “immunogenic polypeptide” and “immunogenic amino acidsequence” refer to a polypeptide or amino acid sequence, respectively,which elicit antibodies that neutralize viral infectivity, and/ormediate antibody-complement or antibody dependent cell cytotoxicity toprovide protection of an immunized host. An “immunogenic polypeptide” asused herein, includes the full length (or near full length) sequence ofthe desired protein or an immunogenic fragment thereof.

By “immunogenic fragment” is meant a fragment of a polypeptide whichincludes one or more epitopes and thus elicits antibodies thatneutralize viral infectivity, and/or mediates antibody-complement orantibody dependent cell cytotoxicity to provide protection of animmunized host. Such fragments will usually be at least about 5 aminoacids in length, and preferably at least about 10 to 15 amino acids inlength. There is no critical upper limit to the length of the fragment,which could comprise nearly the fill length of the protein sequence, oreven a fusion protein comprising fragments of two or more of theantigens.

By “infectious” is meant having the capacity to deliver the viral genomeinto cells.

A “substantially pure” protein will be free of other proteins,preferably at least 10% homogeneous, more preferably 60% homogeneous,and most preferably 95% homogeneous.

BRIEF DESCRIPTION OF THE DRAWING FIGURE

FIG. 1 is a diagram of BAV-1 genomic DNA showing the relative size ofvarious regions in kilobase pairs. Fragments are lettered in order ofdecreasing size.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various publications, patents and patentapplications are referenced. The disclosures of these publications,patents and patent applications are herein incorporated by reference.

The methods and compositions of the present invention involve modifyingDNA sequences from various prokaryotic and eucaryotic sources and bygene insertions, gene deletions, single or multiple base changes, andsubsequent insertions of these modified sequences into the genome of anadenovirus. One example includes inserting parts of an adenovirus DNAinto plasmids in bacteria, reconstructing the virus DNA while in thisstate so that the DNA contains deletions of certain sequences, and/orfurthermore adding foreign DNA sequences either in place of thedeletions or at sites removed by the deletions.

Generally, the foreign gene construct is cloned into an adenovirusnucleotide sequence which represents only a part of the entireadenovirus genome, which may have one or more appropriate deletions.This chimeric DNA sequence is usually present in a plasmid which allowssuccessful cloning to produce many copies of the sequence. The clonedforeign gene construct can then be included in the complete viralgenome, for example, by in vivo recombination following a DNA-mediatedcotransfection technique. Multiple copies of a coding sequence or morethan one coding sequences can be inserted into the viral genome so thatthe recombinant virus can express more than one foreign protein ormultiple copies of the same protein. The foreign gene can haveadditions, deletions or substitutions to enhance expression and/orimmunological effects of the expressed protein.

In order for successful expression of the gene to occur, it can beinserted into an expression vector together with a suitable promoterincluding enhancer elements and polyadenylation sequences. A number ofeucaryotic promoter and polyadenylation sequences which providesuccessful expression of foreign genes in mammalian cells and how toconstruct expression cassettes, are known in the art, for example inU.S. Pat. No. 5,151,267. The promoter is selected to give optimalexpression of immunogenic protein which in turn satisfactorily leads tohumoral, cell mediated and mucosal immune responses according to knowncriteria.

The polypeptide encoded by the foreign DNA sequence is produced byexpression in vivo in a recombinant virus-infected cell. The polypeptidemay be immunogenic. More than one foreign gene can be inserted into theviral-genome to obtain successful production of more than one effectiveprotein.

Therefore, one utility of the use of a mutant adenovirus or the additionof a foreign DNA sequence into the genome of an adenovirus is tovaccinate an animal. For example, a mutant virus could be introducedinto an animal to elicit an immune response to the mutant virus.

Alternatively, a recombinant adenovirus having a foreign DNA sequenceinserted into its genome that encodes a polypeptide may also serve toelicit an immune response in an animal to the foreign DNA sequence, thepolypeptide encoded by the foreign DNA sequence and/or the adenovirusitself. Such a virus may also be used to introduce foreign DNA and itsproducts into the host animal to alleviate a defective genomic conditionin the host animal or to enhance the genomic condition of the hostanimal.

While the present invention is not limited to the use of particularviral vectors, in preferred embodiments the present invention utilizesbovine adenovirus expression vector systems. In particularly preferredembodiments, the present invention comprises a bovine adenovirus inwhich part or all of the E4 gene region is deleted and/or into whichforeign DNA is introduced. Alternatively, the system comprises a bovineadenovirus 1 (BAV-1) in which part or all of the E3 and/or E4 generegions are deleted and/or into which foreign DNA is introduced.

The present invention is not limited by the foreign genes or codingsequences (viral, prokaryotic, and eukaryotic) that are inserted into abovine adenovirus nucleotide sequence in accordance with the presentinvention. Typically the foreign DNA sequence of interest will bederived from pathogens that in bovine cause diseases that have aneconomic impact on the cattle or dairy industry. The genes may bederived from organisms for which there are existing vaccines, andbecause of the novel advantages of the vectoring technology, theadenovirus derived vaccines will be superior. Also, the gene of interestmay be derived from pathogens for which there is currently no vaccinebut where there is a requirement for control of the disease. Typically,the gene of interest encodes immunogenic polypeptides of the pathogenand may represent surface proteins, secreted proteins and structuralproteins.

The present invention is not limited by the particular organisms fromwhich a foreign DNA sequence is obtained for gene insertion into abovine adenovirus genome. In preferred embodiments, the foreign DNA isfrom bovine rotavirus, bovine coronavirus, bovine herpes virus type 1,bovine respiratory syncytial virus, bovine para influenza virus type 3(BPI-3), bovine diarrhea virus, bovine rhinotracheitis virus, bovineparainfluenza type 3 virus, Pasteurella haemolytica, Pasteurellamultocida and/or Haemophitus somnus. In another preferred embodiment,the foreign DNA encodes a cytokine.

The present invention is also not limited to the use of a particular DNAsequence from such an organism. Often selection of the foreign DNAsequence to be inserted into an adenovirus genome is based upon theprotein it encodes. Preferably, the foreign DNA sequence encodes animmunogenic polypeptide.

The preferred immunogenic polypeptide to be expressed by the virussystems of the present invention contain full-length (or nearfull-length) sequences encoding antigens. Alternatively, shortersequences that are immunogenic (i.e., encode one or more epitopes) canbe used. The shorter sequence can encode a neutralizing epitope, whichis defined as an epitope capable of eliciting antibodies that neutralizevirus infectivity in an in vitro assay. Preferably the peptide shouldencode a protective epitope that is capable of raising in the host anprotective immune response; i.e., an antibody-mediated and/or acell-mediated immune response that protects an immunized host frominfection. In some cases the gene for a particular antigen can contain alarge number of introns or can be from an RNA virus. In these cases acomplementary DNA copy (cDNA) can be used.

It is also possible to use fragments of nucleotide sequences of genesrather than the complete sequence as found in the wild-type organism.where available, synthetic genes or fragments thereof can also be used.However, the present invention can be used with a wide variety of genesand/or fragment and is not limited to those set out herein.

Thus, the antigens encoded by the foreign DNA sequences used in thepresent invention can be either native or recombinant immunogenicpolypeptides or fragments. They can be partial sequences, full-lengthsequences, or even fusions (e.g., having appropriate leader sequencesfor the recombinant host and/or with an additional antigen sequence foranother pathogen).

The present invention is also not limited by the ability of theresulting recombinant and mutant viruses to replicate. In a preferredembodiment, the mutant and recombinant viruses of the present inventionare replication competent. In this manner, a complimenting cell line isnot necessary to produce adequate supplies of virus.

As stated above, the present invention contemplates the administrationof the recombinant and mutant viruses of the present invention tovaccinate an animal. The present invention is not limited by the natureof administration to an animal. For example, the antigens used in thepresent invention, particularly when comprised of short oligopeptides,can be conjugated to a vaccine carrier. Vaccine carriers are well knownin the art: for example, bovine serum albumin (BSA), human serum albumin(HSA) and keyhole limpet hemocyanin (KLH). A preferred carrier protein,rotavirus VP6, is disclosed in EPO Pub. No. 0259149.

The vaccines of the present invention carrying foreign genes orfragments can also be orally administered in a suitable oral carrier,such as in an enteric-coated dosage form. Oral formulations include suchnormally-employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharincellulose, magnesium carbonate, etc. Oral vaccine compositions may betaken in the form of solutions (e.g., water), suspensions, tablets,pills, capsules, sustained release formulations, or powders, containingfrom about 10% to about 95% of the active ingredient, preferably about25% to about 70%. An oral vaccine may be preferable to raise mucosalimmunity in combination with systemic immunity, which plays an importantrole in protection against pathogens infecting the gastrointestinaltract.

In addition, the vaccine can be formulated into a suppository Forsuppositories, the vaccine composition will include traditional bindersand carriers, such as polyalkaline glycols or triglycerides. Suchsuppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), preferablyabout 1% to about 2%.

Protocols for administering the vaccine composition(s) of the presentinvention to animals are within the skill of the art in view of thepresent disclosure. Those skilled in the art will select a concentrationof the vaccine composition in a dose effective to elicit an antibodyand/or T-cell mediated immune response to the antigenic fragment.

The timing of administration may also be important. For example, aprimary inoculation preferably may be followed by subsequent boosterinoculations if needed. It may also be preferred, although optional, toadminister a second, booster immunization to the animal several weeks toseveral months after the initial immunization. To insure sustained highlevels of protection against disease, it may be helpful to readministera booster immunization to the animals at regular intervals, for exampleonce every several years. Alternatively, an initial dose may beadministered orally followed by later inoculations, or vice versa.Preferred vaccination protocols can be established through routinevaccination protocol experiments.

The dosage for all routes of administration of an in vivo recombinantvirus vaccine depends on various factors, including the size of thepatient, the nature of the infection against which protection is needed,the type of carrier and other factors, which can readily be determinedby those of skill in the art. By way of non-limiting example, a dosageof between 10³ plaque forming units (pfu) and 10⁸ pfu can be used.

The present invention also includes a method for providing gene therapyto a mammal in need thereof to control a gene deficiency. In oneembodiment, the methods comprises administering to said mammal a liverecombinant bovine adenovirus containing a foreign nucleotide sequenceencoding a non-defective form of a gene. The foreign nucleotide sequenceis either incorporated into the mammalian genome or is maintainedindependently to provide expression of the required gene in the targetorgan or tissue. These kinds of techniques have recently been used bythose of skill in the art to replace a defective gene or portion thereofFor example, U.S. Pat. No. 5,399,346 to Anderson et al. describestechniques for gene therapy. Moreover, examples of foreign genesnucleotide sequences or portions thereof that can be incorporated foruse in a conventional gene therapy include, but are not limited to,cystic fibrosis transmembrane conductance regulator gene, humanminidystrophin gene, alpha 1-antitrypsin gene and others.

Methods for constructing, selecting and purifying recombinant adenovirusare detailed below in the materials, methods and examples below. Thefollowing serve to illustrate certain preferred embodiments and aspectsof the present invention and are not to be construed as limiting thescope thereof.

Preparation of Bovine Adenovirus (BAV-1) Stock

Bovine adenovirus stocks were prepared by infecting tissue culturecells, Madin-Darby bovine kidney cells (MDBK), at a multiplicity ofinfection of 0.01 PFU/cell in Dulbecco's Modified Eagle Medium (DMEM)containing 2 mM glutamine, 100 units/ml penicillin, 100 units/mlstreptomycin (these components are obtained from Sigma (St. Louis, Mo.)or an equivalent supplier, and hereafter are referred to as complete DMEmedium) plus 1% fetal bovine serum. After cytopathic effect wascomplete, the medium and cells were harvested. After one or two cyclesof freezing (−70° C.) and thawing (37° C.), the infected cells werealiquot as 1 ml stock and stored frozen at −70″C.

Preparation of Bovine Adenovirus (BAV-1) DNA

All manipulations of bovine adenovirus were made using strain 10 (ATCCVR-313). For the preparation of BAV-1 viral DNA from the cytoplasm ofinfected cells, MDBK cells were infected at a multiplicity of infection(MOI) sufficient to cause extensive cytopathic effect before the cellsovergrew. All incubations were carried out at 37° C. in a humidifiedincubator with 5% CO₂ in air.

The best DNA yields were obtained by harvesting monolayers which weremaximally infected, but showing incomplete cell lysis (typically 5-7days). Infected cells were harvested by scraping the cells into themedium using a cell scraper (Costar brand). The cell suspension wascentrifuged at 3000 rpm for 10 minutes at 5° C. in a GS-3 rotor (SorvallInstruments, Newtown, Conn.). The resultant pellet was resuspended incold PBS (20 ml/Roller Bottle) and subjected to another centrifugationfor 10 minutes at 3000 rpm in the cold.

After decanting the PBS, the cellular pellet was resuspended in 5ml/roller bottle of TE buffer (10 mM Tris pH 7.5 and 1 mM EDTA) andswell on ice for 15 minutes. NP40 Nonidet P40.™.; Sigma, St. Louis, Mo.)was added to the sample to a final concentration of 0.5% and keep on icefor another 15 minutes. The sample was centrifuged for 10 minutes at3000 rpm in the cold to pellet the nuclei and remove cellular debris.

The supernatant fluid was carefully transferred to a 30 ml Corexcentrifuge tube. SDS (sodium dodecyl sulfate; stock 20%) were added tothe sample to final concentrations of 1%. 200 μl of proteinase-K at 10mg/ml (Boelringer Mannheim, Indianapolis, Ind.) was added per rollerbottle of sample, mixed, and incubated at 45° C. for 1-2 hours.

After this period, an equal volume of water-saturated phenol was addedto the sample and mixed by vortex. The sample was spun in a clinicalcentrifuge for 5 minutes at 3000 rpm to separate the phases. NaAc wasadded to the aqueous phase to a final concentration of 0.3M (stocksolution 3M pH 5.2), and the nucleic acid precipitated at −70° C. for 30minutes after the addition of 2.5 volumes of cold absolute ethanol. DNAin the sample was pelleted by spinning for 20 minutes to 8000 rpm in anHB-4 rotor at 4° C.

The supernatant was carefully removed and the DNA pellet washed oncewith 25 ml of 80% ethanol. The DNA pellet was dried briefly by vacuum(2-3 minutes), and resuspended in 2 ml/roller bottle of infected cellsof TE buffer (20 mM Tris pH 7.5, 1 mM EDTA). 10 μl of RNaseA at 10 mg/ml(Sigma, St. Louis, Mo.) was added and incubate at 37° C. for one hour.0.5 ml of 5N NaCl and 0.75 ml of 30% PEG was added and precipitated at4° C. overnight.

DNA in the sample was pelleted by spinning for 20 minutes to 8000 rpm inan HB-4 rotor at 4° c. Resuspend pellet in 2 ml TE buffer (20 mM TrispH7.5, 1 mM EDTA and 0.2% SDS) and extracted with an equal volume ofwater-saturated phenol. The sample was spun in a clinical centrifuge for5 minutes at 3000 rpm to separate the phases. NaAc was added to theaqueous phase to a final concentration of 0.3M (stock solution 3M pH5.2), and the nucleic acid precipitated at −70° C. for 30 minutes afterthe addition of 2.5 volumes of cold absolute ethanol.

DNA in the sample was pelleted by spinning for 20 minutes to 8000 rpm inan HB-4 rotor at 5° C. The supernatant was carefully removed and the DNApellet washed once with 25 ml of 80% ethanol. The DNA pellet was driedbriefly by vacuum (2-3 minutes), and resuspended in 200 μl/roller bottleof infected cells of TE buffer (10 mM Tris pH 7.5, 1 mM EDTA). All viralDNA was stored at approximately 4° C.

Molecular Biological Techniques

Techniques for the manipulation of bacteria and DNA, including suchprocedures as digestion with restriction endonucleases, gelelectrophoresis, extraction of DNA from gels, ligation, phosphorylationwith kinase, treatment with phosphatase, growth of bacterial cultures,transformation of bacteria with DNA, and other molecular biologicalmethods are described by Maniatis et al. (T. Maniatis, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982)) andSambrook et al. (J. Sambrook, et al., Molecular Cloning: A LaboratoryManual Second Edition, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989)). The polymerase chain reaction (PCR) was used to introducerestriction sites convenient for the manipulation of various DNAs. Theprocedures used are described by Innis et al (M. A. Innis, et al., PCRProtocols: A Guide To Methods And Applications, pp. 84-91, AcademicPress, Inc., San Diego, Calif. (1990)).

In general, amplified fragments were less than 2000 base pairs in sizeand critical regions of amplified fragments were confirmed by DNAsequencing. Except as noted, these techniques were used with minorvariations.

DNA Sequencing

DNA sequencing was performed on the Applied Biosystems AutomatedSequencer Model 373A (with XL upgrade) per instructions of themanufacturer. Subclones were made to facilitate sequencing. Internalprimers were synthesized on an ABI 392 DNA synthesizer or obtainedcommercially (Genosys Biotechnologies, Inc., The Woodlands, Tex.) largerDNA sequences were built utilizing consecutive overlapping primers.Sequence across the junctions of large genomic subclones was determineddirectly using a full length genomic clone as template. Assembly,manipulation and comparison of sequences was performed with DNAstarprograms. Comparisons with GenBank were performed using NCBI BLASTprograms (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schäffer,Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997),“Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms”, Nucleic Acids Res. 25:3389-3402.).

Construction of Recombinant BAV-1 Genomes in E. coli

Recombinant BAV-1 genomes are constructed by homologous recombinationaccording to the method of C. Chartier et al (1996) J. of Virology70:805-4810.

A small, easily manipulated plasmid was constructed containingapproximately 1000 base pairs each of the Left and Right ends of theBAV-1 genome. Homologous recombination between this vector and BAV-1genomic DNA results in a plasmid containing the entire BAV-1 genome(adenoviral backbone vector). This BAV-1 genomic plasmid may be used togenerate recombinant genomes by linearization of the plasmid andrecombination with homology DNAs engineered to contain foreign DNAflanked by DNA derived from the desired BAV-1 insertion site. Note thatin order to linearize the adenoviral backbone vector, an infrequentcutting enzyme must be located within the region analogous to theflanking BAV-1 sequences.

We have mapped the restriction sites of such an enzyme. PvuI cuts theBAV-1 genome at two locations one in the BamH1 D fragment and one in theBamH1 C fragment (see FIG. 1). The adenoviral backbone vector contains athird PvuI site within the antibiotic resistance gene of the plasmid.The PvuI site within the BamH1 C fragment is suitable for gene insertionsites within both the E3 and E4 regions. Therefore a partial PvuIdigestion of the adenoviral backbone vector will yield a sub populationof molecules linearized at the PvuI site in the BamH1 C fragment. Thesemolecules may recombine with the homology DNA to generate a viableplasmid. Molecules linearized at the other two sites will not be able torecombine to generate viable plasmids.

The high competence of bacteria cells E. coli BJ5183 recBC sbcBC (D.Hanahan (1983) J. Mol. Biol. 166:557-580) is desired to achieveefficient recombination. Typically, 10 nanograms of a restrictionfragment containing foreign DNA flanked by the appropriate BAV insertionsequences (homology DNA) is mixed with 1 nanogram of linearizedadenoviral backbone vector in a total volume of 10 μl. Fifty microlitersof competent BJ5183 cells were added. After 15 min. on ice, 5 min. at37° C. and 15 min. on ice, 200 μl of LB was added and the cells platedon agar containing LB+80 μg/ml carbenicillin, after one hour at 37° C.,

Low temperature (25-27° C.) for growing small scale cultures (forscreening carbenicillin resistant colonies) and subsequent large scalecultures (for isolation of large quantities of plasmid DNA) isessential. Carb^(R) colonies were first grown in 4-5 ml cultures at25-27° C. for two days. Small scale DNAs were prepared using boilingmethod (J. Sambrook, et al., Molecular Cloning: A Laboratory ManualSecond Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989)) and analyzed by DNA restriction analysis. To purify the DNA awayfrom vector DNA concatemers the UNAs from correct clones werere-transform into DH10B (Life Technologies) cells. Analysis of bacterialcolonies by DNA restriction analysis was repeated. Glycerol stocks wereprepared from the correct clones and stored at −70° C. Large quantitiesof plasmid DNA were prepared using Qiagen Plasmid Kit (Qiagen Inc.) orscale-up of boiling method from 250 ml cultures which were inoculatedwith glycerol stock and grown at 37° C. for one day.

Transfection of BAV-1 DNA

Approximately 1.5×10⁵ cells/ml (MBK) were plated in 6 cm plates 24 hrbefore transfection, by which time they reached 50-70% confluency. Fortransfection the Lipofectin method was used according to themanufacturer's instructions (Lipofectin, Life Technologies, Rockville,Md.). A transfection mix was prepared by adding several (4-15) μg ofBAV-1 viral DNA or linearized genome plasmid DNA and 50 μl of LipofectinReagent to 200 μl of serum-free medium according to the manufacturer'sinstruction.

After incubation at room temperature for 15-30 min, the transfection mixwas added to the cells. After 4-6 hr at 37° C., the media containing thetransfection mix was removed, and 5 ml of growth medium was added.Cyopathic effect became apparent within 7-10 days. The transfected virusstock was harvested by scraping cells in the culture and stored at −70°C.

Plaque Purification of Recombinant Constructs

Monolayers of MDBK cells in 6 cm or 10 cm plates were infected withtransfection stock, overlaid with nutrient agarose media and incubatedfor 5-10 days at 37° C. Once plaques have developed, single andwell-isolated plaque was picked onto MDBK cells. After 5-10 days when80-90% cytopathic effect was reached, the infected cells (P1 stock) wereharvested and stored at −70° C. This procedure was repeated one moretime with P1 stock.

Cloning of Bovine Viral Diarrhea Virus (BVDV) Glycoprotein 53 (g53) Gene

The bovine viral diarrhea g53 gene was cloned by a PCR cloning procedureessentially as described by Katz et al. (Journal of Virology 64:1808-1811 (1990)) for the HA gene of human influenza. Viral RNA preparedfrom BVD virus Singer strain grown in MDBK cells was first converted tocDNA utilizing an oligonucleotide primer specific for the target gene.The cDNA was then used as a template for polymerase chain reaction (PCR)cloning (M. A. Innis et al., PCR Protocols: A Guide to Methods andApplications, 84-91, Academic Press, Inc. San Diego (1990)) of thetargeted region. The PCR primers were designed to incorporaterestriction sites which permit the cloning of the amplified codingregions into vectors containing the appropriate signals for expressionin BAV-1. One pair of oligonucleotides were required for the codingregion. The g53 gene coding region (amino acids 1-394) from the BVDVSinger strain (M. S. Collett et al., Journal of Virology 65, 200-208,(1988)) was cloned using the following primers:5′-CTGGATCCTCATCCATACTGAGTCCCTGAGGCCTTCTGTTC-3′ [SEQ ID NO: 1] for cDNApriming and combined with 5′-CATAGATCTTGTGGTGCTGTCCGACTT CGCA-3′ [SEQ IDNO: 2] for PCR.

Western Blotting Procedure

Samples of lysates and protein standards were on a polyacrylamide gelaccording to the procedure of Laemnli, Nature 227, 680-685 (1970)).After gel electrophoresis the proteins were transferred and processedaccording to Sambrook, et al., Molecular Cloning A Laboratory ManualSecond Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989). The primary antibody was a mouse monoclonal antibody (Mab 6.12.2from Dubovi, Cornell University, Ithaca, N.Y.) diluted 1:100 with 5%non-fat dry milk in Tris-sodium chloride, and sodium Azide (TSA: 6.61gTris-HCl, 0.97 g Tris-base, 9.0 g NaCl and 2.0 g Sodium Azide per literH2O). The secondary antibody was a goat anti-mouse alkaline phosphataseconjugate diluted 1:1000 with TSA.

Plasmid 990-11

Plasmid 990-11 contains the Left and Right ends of the BAV-1 genome.These sequences were cloned by standard PCR methods (M. A. Innis, etal., PCR Protocols: A Guide To Methods And Applications, pp. 84-91,Academic Press, Inc., San Diego, Calif. (1990) using primers based onthe sequences determined for the EcoR1 A and BamH1 F fragmentsrespectively. Primers (1) 5′-3′ GGCCTTAATTAACATCATCAATAATA TACGGAACAC[SEQ ID NO: 5] and (2) 5′-3′ GGAAGATCTTGAGCATGCAGAGC AATTCACGCCGGGTAT[SEQ ID NO: 6] were used to PCR the Left end of BAV-1. Since threerepetitive elements within this region shared the same 5′ end sequences(i. e. primer 1), 1760 bp, 1340 bp and 920 bp BAV-1 DNA fragments wereamplified by PCR. The 920 bp DNA fragment was cloned into PCR-Bluntvector (Invitrogen, Carlsbad, Calif.). Primers (1) and (3)5′-GGCAATGAGATCTTTTGGATGACAAGCTGAGCTACGCG-3′ [SEQ ID NO: 7] were used toPCR the Right end of BAV-1, 740 bp and 1160 bp PCR products wereamplified and 1160 bp fragment DNA was cloned into pCR-Blunt vector(Invitrogen, Carlsbad, Calif.). Plasmid 990-11 was then constructed bycloning the BAV-1 end fragments into the polylinker of plasmid pPolyII(R. Lathe, J. L. Vilotte, and A. J. Clark, Gene, 57:193-201, 1997). Theend fragments were cloned as a single PacI fragment containing a uniqueBglII site at their internal junction. Only the BamH1 and EcoR1 siteswere retained from the polylinker.

Plasmid 990-50 (Adenoviral Backbone Vector)

Plasmid 990-50 was constructed according to the method described above(Construction of Recombinant BAV-1 Genomes in E. coli). Briefly,co-transformation of the BglII-linearized plasmid 990-11 and BAV-1genomic DNA regenerated a stable circular plasmid containing the entireBAV-1 genome. In this plasmid PacI sites flank the inserted BAV-1genomic sequences. As Pac is absent from BAV-1 genomic DNA, digestionwith this enzyme allows the precise excision of the full-length BAV-1genome from the plasmid 990-50.

Plasmid 996-80D

Plasmid 996-80D contains DNA encompassing approximately 5945 base pairsof the Right end of the BAV-1 genome from which the EcoR1 “G” and “H”fragments have been deleted and replaced with a synthetic SmaI site. Theplasmid was constructed for the purpose of deleting a portion of theBAV-1 E4 region. It may also be used to insert foreign DNA intorecombinant BAV-1 genomes. It contains a unique SmaI restriction enzymesite into which foreign DNA may be inserted. The plasmid may beconstructed utilizing standard recombinant DNA techniques (see aboveMolecular Biological Techniques) by joining restriction fragments fromthe following sources with the synthetic DNA sequences indicated. Theplasmid vector is derived from an approximately 2774 base pair HindIIIto PvuII restriction fragment of pSP64 (Promega Corporation, Madison,Wis.). The synthetic linker sequence 5′-CTGTAGATCTGCGGCCGCGTTTAAACGTCGACAAGCTTCCC-3′ [SEQ ID NO: 8] is ligated to the PvuII site of pSP64(Promega Corporation, Madison, Wis.). Fragment 1 is an approximately1693 base pair PstI to EcoR1 sub fragment of the BAV-1 BamH1 “C”fragment (positions 28241 to 2993 from SEQ ID NO: 3). The syntheticlinker sequence 5′-AATTCGAGCTCGCCCGGGCGAGCTCGA-3′ [SEQ ID NO: 9] isligated to fragment 1 retaining EcoR1 sites at both ends of the linkersequence. Fragment 2 is an approximately 48 base pair EcoR1 to BamH1restriction sub fragment of the BAV-1 BamH1 “C” fragment positions 31732to 31779 from SEQ ID NO: 3). Fragment 3 is the approximately 2406 basepair BAV-1 BamH1 “F” fragment positions 31780 to 34185 from SEQ ID NO:3). The synthetic linker sequence5′-GACTCTAGGGGCGGGGAGTTTAAACGCGGCCGCAGATCTAGCT-3′ [SEQ ID NO: 10] isligated between fragment 3 and the HindIII site of pSP64 (PromegaCorporation, Madison, Wis.). Note that the BAV-1 sequences can be cutout of this plasmid via the NotI restriction sites located in theflanking synthetic linker sequences.

Plasmid 1004-73.16.14

Plasmid 1004-73.16.14 contains a recombinant BAV-1 genome from which theEcoR1 “G” and “H” fragments have been deleted and replaced by asynthetic SmaI site (5′-GAATTCGAGCTCGCCCGGGCGAGCTCGAATTC-3′) [SEQ ID NO:11]. This plasmid may be used to generate recombinant bovine adenovirusvectors with deletion and gene insertions at the E4 region. The plasmidmay be constructed according to the method above (Construction ofRecombinant BAV-1 Genomes in E. coli). The homology DNA is derived fromthe NotI insert of plasmid 996-80D and the adenoviral backbone vectorplasmid 990-50 is linearized by partial digestion with the PvuI.

Plasmid 1004-07.16

Plasmid 1004-07.16 was constructed by inserting a BVDV g53 gene,engineered to be under control of the human cytomegalovirus immediateearly promoter (Invitrogen, Carlsbad, Calif.), into the unique SmaI siteof plasmid 996-80D. The BVDV g53 gene was isolated according to themethod above (Cloning of Bovine Viral Diarrhea virus g53 gene).

Plasmid 1004-40

Plasmid 1004-40 contains a recombinant BAV-1 genome from which the EcoR1G and H fragments have been deleted, The gene for the bovine viraldiarrhea virus (BVDV) glycoprotein 53 (g53) (amino acids 1-394) underthe control of the HCMV immediate early promoter was inserted into thedeleted region. The plasmid may be constructed according to the methodabove (Construction of Recombinant BAV-1 Genomes in E. coli). Thehomology DNA is derived from the NotI insert of plasmid 1004-17.16 andthe adenoviral backbone vector plasmid 990-50 is linearized by partialdigestion with the PvuI.

Plasmid 1018-14.2

Plasmid 1018-14.2 contains DNA flanking the E3 region of BAV-1, fromwhich a specific region of this sequence flanked by SalI and BamH1 sites(positions 25664 to 26840 from SEQ ID NO: 3) has been deleted. Theplasmid was constructed for the purpose of deleting the correspondingportion of the BAV-1 E3 region. It may also be used to insert foreignDNA into recombinant BAV-1 genomes. It contains a unique HindIIIrestriction enzyme site into which foreign DNA may be inserted. Theplasmid may be constructed utilizing standard recombinant DNA techniques(see above Molecular Biological Techniques) by joining restrictionfragments from the following sources with the synthetic DNA sequencesindicated. The plasmid vector is derived from an approximately 2774 basepair HindIIII to PvuII restriction fragment of pSP64 (PromegaCorporation, Madison, Wis.). The synthetic linker sequence5′-CTGTAGATCTG CGGCCGCGTTTAAACG-3′ [SEQ ID NO: 12] is ligated to thePvuII site of pSP64 (Promega Corporation, Madison, Wis.). Fragment 1 isan approximately 2665 base pair SalI to SalI sub fragment (positions22999 to 25663 from SEQ ID NO: 3) of the BAV-1 BamH1 B fragment.Fragment 1 is ligated to the upstream synthetic sequence retaining theSalI site at the junction. Fragment 1 contains a unique AvaI site(positions 25317 to 25322 from SEQ ID NO: 3). Fragment 1 is orientedsuch that the unique AvaI site is closer (406 base pairs) to fragment 2than to the plasmid vector. The synthetic linker sequence5′-TCGACAAGCTTCCC-3′ [SEQ ID NO: 13] is ligated to second end offragment 1 again retaining the SalI site at the junction. Fragment 2 isan approximately 4223 base pair BamH1 to HindIII restriction subfragment of the BAV-1 BamH1 C fragment (positions 26851 to 31073 fromSEQ ID NO: 3). Note that the end of both fragments were blunt end bytreatment with T4 polymerase. The synthetic linker sequence 5′-CCCGGGAGTTTAAACGCGGCCGCAGATCTAGCT-3′ [SEQ ID NO. 14] is ligated betweenfragment 2 and the HindIII site of pSP⁶⁴ (Promega Corporation, Madison,Wis.). Note that the HindIII site is not retained. The BAV-1 sequencescan be cut out of this plasmid via the NotI restriction sites located inthe flanking synthetic linker sequences.

Plasmid 1018-75

Plasmid 1018-75 contains a recombinant BAV-1 genome from which aspecific region of the BamH1 “B” fragment (positions 25664 to 26840 fromSEQ ID NO: 3) has been deleted. This plasmid may be used to generaterecombinant bovine adenovirus vectors with deletions and gene insertionsat the E3 region. The plasmid may be constructed according to the methodabove (Construction of Recombinant BAV-1 Genomes in E. coli). Thehomology DNA is derived from the NotI insert of plasmid 1018-14.2 andthe adenoviral backbone vector plasmid 990-50 is linearized by partialdigestion with the PvuI.

Plasmid 1018-23C15

Plasmid 1018-23C15 was constructed by inserting a BVDV g53 gene,engineered to be under control of the human cytomegalovirus immediateearly promoter (Invitrogen, Carlsbad, Calif.), into the unique HindIIIsite of plasmid 1018-14.2. The BVDV g53 gene was isolated according tothe method above (Cloning of Bovine Viral Diarrhea virus g53 gene).

Plasmid 1018-42

Plasmid 1018-42 contains a recombinant BAV-1 genome from which aspecific region of the BamH1 “B” fragment (positions 25664 to 26840 fromSEQ ID NO: 3) has been deleted. The gene for the bovine viral diarrheavirus (BVDV) glycoprotein 53 (g53) (amino acids 1-394) under the controlof the HCMV immediate early promoter was inserted into the deletedregion. The plasmid may be constructed according to the method above(Construction of Recombinant BAV-1 Genomes in E. coli). The homology DNAis derived from the NotI insert of plasmid 1018-23C15 and the adenoviralbackbone vector plasmid 990-50 is linearized by partial digestion withthe PvuI.

Plasmid 1018-45

Plasmid 1018-45 contains DNA flanking the E3 region of BAV-1, from whicha specific region of this sequence flanked by EcoR1 and BamH1 sitespositions 25765 to 26850 from SEQ ID NO: 3) has been deleted. Theplasmid was constructed for the purpose of deleting the correspondingportion of the BAV-1 E3 region. It may also be used to insert foreignDNA into recombinant BAV-1 genomes. It contains a unique HindIIIrestriction enzyme site into which foreign DNA may be inserted. Theplasmid may be constructed utilizing standard recombinant DNA techniques(see above Molecular Biological Techniques) by joining restrictionfragments from the following sources with the synthetic DNA sequencesindicated. The plasmid vector is derived from an approximately 2774 basepair HindIIII to PvuII restriction fragment of pSP64 (PromegaCorporation, Madison, Wis.). The synthetic linker sequence 5′-CTGTAGATCTGCGGCCGCGTTTAACG-3′ [SEQ ID NO: 12] is ligated to the PvuII site ofpSP64 (Promega Corporation, Madison, Wis.). Fragment 1 is anapproximately 1582 base pair SacI to EcoR1 sub fragment (positions 24183to 25764 from SEQ ID NO: 3) of the BAV-1 BamH1 B fragment. Fragment 1 isligated to the upstream synthetic sequence. The fragment was blunted endwith T4 polymerase treatment so neither the SacI nor EcoR1 sites areretained. Fragment 1 contains a unique AvaI site (positions 25317 to25322 from SEQ ID NO: 3). Fragment 1 is oriented such that the uniqueAvaI site is closer (406 base pairs) to fragment 2 than to the plasmidvector. The synthetic linker sequence 5′-CAAGCTTCCC-3′ [SEQ ID NO: 17]is ligated to second end of fragment 1 again retaining the SalI site atthe junction. Fragment 2 is an approximately 4223 base pair BamH1 toHindIII restriction sub fragment of the BAV-1 BamH1 C fragment positions26851 to 31073 from SEQ ID NO: 3). Note that the end of both fragmentswere blunt end by treatment with T4 polymerase. The synthetic linkersequence 5′-CCCGG GAGTTTAAACGCGGCCGCAGATCTAGCT-3′ [SEQ ID NO: 14] isligated between fragment 2 and the HindIII site of pSP64 (PromegaCorporation, Madison, Wis.). Note that the HindIII site is not retained.The BAV-1 sequences can be cut out of this plasmid via the NotIrestriction sites located in the flanking synthetic linker sequences.

Plasmid 1028-03

Plasmid 1028-03 contains a recombinant BAV-1 genome from which aspecific region of the BamH1 “B” fragment (positions 25765 to 26850 fromSEQ ID NO: 3) has been deleted. This plasmid may be used to generaterecombinant bovine adenovirus vectors with deletions and gene insertionsat the E3 region. The plasmid may be constructed according to the methodabove (Construction of Recombinant BAV-1 Genomes in E. coli). Thehomology DNA is derived from the NotI insert of plasmid 1018-45 and theadenoviral backbone vector plasmid 990-50 is linearized by partialdigestion with the PvuI.

Plasmid 1028-77

Plasmid 1028-77 was constructed by inserting a BVDV g53 gene, engineeredto be under control of the human cytomegalovirus immediate earlypromoter (Invitrogen, Carlsbad, Calif.), into the unique HindIII site ofplasmid 1018-45. The BVDV g53 gene was isolated according to the methodabove (Cloning of Bovine Viral Diarrhea virus g53 gene).

Plasmid 1038-16

Plasmid 1038-16 contains a recombinant BAV-1 The genome from which aspecific region of the BamH1 “B” fragment (positions 25765 to 26850 fromSEQ ID NO: 3) has been deleted. The gene for the bovine viral diarrheavirus (BVDV) glycoprotein 53 (g53) (amino acids 1-394) under the controlof the HCMV immediate early promoter was inserted into the deletedregion. The plasmid may be constructed according to the method above(Construction of Recombinant BAV-1 Genomes in E. coli). The homology DNAis derived from the NotI insert of plasmid 1028-77 and the adenoviralbackbone vector plasmid 990-50 is linearized by partial digestion withthe PvuI.

Plasmid 1054-93

Plasmid 1054-93 contains DNA derived from the E4 region of BAV-1. Thesequence corresponding to positions 33614 to 3375 fro SEQ ID NO: 3 hasbeen deleted and replaced with a synthetic PstI site. The plasmid wasconstructed for the purpose of deleting a portion of the BAV-1 E4region. It may also be used to insert foreign DNA into recombinant BAV-1genomes. It contains a unique PstI restriction enzyme site into whichforeign DNA may be inserted. The plasmid may be constructed utilizingstandard recombinant DNA techniques (see above Molecular BiologicalTechniques) by joining restriction fragments from the following sourceswith the synthetic DNA sequences indicated. Note that fragments derivedfrom BAV-1 DNA are ligated in the orientation indicated by the positionsgiven for each fragment, The plasmid vector is derived from anapproximately 2774 base pair HindIII to PvuII restriction fragment ofpSP64 (Promega Corporation, Madison, Wis.). The synthetic linkersequence 5′-CTGTAGATCTGCGG CCGCGTTTAAACGTCGACAAGCTTCCC-3′ [SEQ ID NO: 8]is ligated to the PvuII site of pSP64 (Promega Corporation, Madison,Wis.). Fragment I is an approximately 3538 base pair PstI to BamHI subfragment of the BAV-1 BamHI “C” fragment positions 28241 to 31779 fromSEQ. ID NO.3. Fragment 1 is ligated to the 3′ end of the syntheticlinker sequence [SEQ ID NO: 8]. Fragment 2 is an approximately 1832 basepair PCR fragment containing sequences derived from the BAV-1 genome(positions 31780 to 33613 from SEQ ID NO: 3). Fragment 2 is ligated tofragment 1 such that the BamHI site at the junction is retained. Thesynthetic linker sequence 5′-CTGCAG-3′ [SEQ ID NO: 4] is ligated tofragment 2. Fragment 3 is an approximately 460 base pair PCR fragmentcontaining sequences derived from the BAV-1 genome positions 33725 to34185 from SEQ ID NO: 3). Fragment 3 is ligated to the 3, end of thesynthetic linker sequence 5′-CTGCAG-3′. The synthetic linker sequence5′-GACTCTAGGGGCGGGGAGTTT AAACGCGGCCGCAGATCTAGCT-3′ [SEQ ID NO: 10] isligated between fragment 3 and the HindIII site of pSP64 (PromegaCorporation, Madison, Wis.). Note that the BAV-1 sequences can be cutout of this plasmid via the NotI restriction sites located in theflanking synthetic linker sequences.

Plasmid 1055-38

Plasmid 1055-38 contains DNA derived from the E4 region of BAV-1. Thesequence encoding nORF13 (see Table 1) has been deleted and replacedwith a synthetic PstI site. The plasmid was constructed for the purposeof deleting a portion of the BAV-1 E4 region. It may also be used toinsert foreign DNA into recombinant BAV-1 genomes. It contains a uniquePstI restriction enzyme site into which foreign DNA may be inserted. Theplasmid may be constructed utilizing standard recombinant DNA techniques(see above Molecular Biological Techniques) by joining DNA fragmentsfrom the following sources with the synthetic DNA sequences indicated.Note that fragments derived from BAV-1 DNA are ligated in theorientation indicated by the positions given for each fragment. Theplasmid vector is derived from an approximately 2774 base pair HindIIIto PvuII restriction fragment of pSP64 (Promega Corporation, Madison,Wis.). The synthetic linker sequence5′-CTGTAGATCTGCGGCCGCGTTTAAACGTCGACAAGCTTCCC-3′ [SEQ ID NO: 8] isligated to the PvuII site of pSPM (Promega Corporation, Madison, Wis.).Fragment 1 is an approximately 1282 base pair PCR fragment containingsequences derived from the BAV-1 genome (positions 28240 to 29522 fromSEQ ID NO: 3). Fragment 1 is ligated to the 3′ end of the syntheticlinker sequence indicated above [SEQ ID NO: 8]. The synthetic linkersequence 5′-CTGCAG-3′ [SEQ ID NO: 4] is ligated to fragment 1. Fragment2 is an approximately 1372 base pair PCR fragment containing sequencesderived from the BAV-1 genome positions 30407 to 31779 from SEQ ID NO:3). Fragment 2 is ligated to the 3′ end of the synthetic linker sequence5′-CTGCAG-3′ [SEQ ID NO: 4]. Fragment 3 is the approximately 2406 basepair BAV-1 BamH1 “F” fragment (positions 31779 to 34185 from SEQ ID NO:3). Fragment 3 is ligated to the 3′ end of fragment 2. The syntheticlinker sequence 5′-GACTCTAGGGGCGGGGAGTTTTA AACGCGGCCGCAGATCTAGCT-3′ [SEQID NO: 10] is ligated between fragment 3 and the HindIII site of SP64(Promega Corporation, Madison, Wis.). Note that the BAV-1 sequences canbe cut out of this plasmid via the NotI restriction sites located in theflanking synthetic linker sequences.

Plasmid 1055-52

Plasmid 1055-52 contains a recombinant BAV-1 genome from which a portionof the E4 region positions 29522-30407 from SEQ ID NO: 3) has beendeleted and replaced by a synthetic PstI site (5-CTGCAG-3′) [SEQ ID NO:4]. This plasmid may be used to generate recombinant bovine adenovirusvectors with gene insertions and/or a deletion at the E4 region. Theplasmid may be constructed according to the method above (Constructionof Recombinant BAV-1 Genomes in E. coli). The homology DNA is derivedfrom the NotI insert of plasmid 1055-38 and the adenoviral backbonevector plasmid 990-50 is linearized by partial digestion with the PvuI.

Plasmid 1055-47

Plasmid 1055-47 was constructed by inserting a BVDV g53 gene into theunique Pst1 site of plasmid 1055-38. The BVDV coding region was insertedin the reverse complimentary orientation such that it is transcribed bythe E4 region promoter located at the right end of the genome. The BVDVg53 gene was isolated according to the method above (Cloning of BovineViral Diarrhea virus g53 gene).

Plasmid 1055-56

Plasmid 1055-56 contains a recombinant BAV-1 genome from which theBAV-1's sequence from positions 29522 to 30407 [SEQ ID NO: 3] has beendeleted. The gene for the BVDV g53 (amino acids 1-394) was inserted intothe deleted region. The plasmid may be constructed according to themethod above (Construction of Recombinant BAV-1 Genomes in E. coli). Thehomology DNA is derived from the NotI insert of plasmid 1055-47 and theadenoviral backbone vector plasmid 990-50 is linearized by partialdigestion with the PvuI.

Plasmid 1055-93

Plasmid 1055-93 contains DNA derived from the E4 region of BAV-1. Thesequence encoding nORF13 (see Table 1) has been deleted and replacedwith a synthetic PstI site. Te plasmid was constructed for the purposeof deleting a portion of the BAV-1 E4 region. It may also be used toinsert foreign DNA into recombinant BAV-1 genomes. It contains a uniquePstI restriction enzyme site into which foreign DNA may be inserted. Theplasmid may be constructed utilizing standard recombinant DNA techniques(see above Molecular Biological Techniques) by joining DNA fragmentsfrom the following sources with the synthetic DNA sequences indicated.Note that fragments derived from BAV-1 DNA are ligated in theorientation indicated by the positions given for each fragment. Theplasmid vector is derived from an approximately 2774 base pair HindIIIto PvuII restriction fragment of pSP64 (Promega Corporation, Madison,Wis.). The synthetic linker sequenceS′-CTGTAGATCTGCGGCCGCGTTTAAACGTCGACAAGCTTCCC-3′ [SEQ ID NO: 8] isligated to the PvuII site of pSP64 (Promega Corporation, Madison, Wis.).Fragment 1 is an approximately 1282 base pair PCR fragment containingsequences derived from the BAV-1 genome positions 28240 to 29522 fromSEQ ID NO: 3). Fragment 1 is ligated to the 3′ end of the syntheticlinker sequence indicated above [SEQ ID NO: 8]. The synthetic linkersequence 5′-CTGCAG-3′ [SEQ ID NO: 4] is ligated to fragment 1. Fragment2 is an approximately 1372 base pair PCR fragment containing sequencesderived from the BAV-1 genome (positions 30403 to 31779 from SEQ ID NO:3). Fragment 2 is ligated to the 3′ end of the synthetic linker sequence5′-CTGCAG-3′ [SEQ ID NO. 4]. Fragment 3 is the approximately 2406 basepair BAV-1 BamH1 “F” fragment (positions 31779 to 34185 from SEQ ID NO:3). Fragment 3 is ligated to the 3′ end of fragment 2. The syntheticlinker sequence 5′-GACTCTAGGGGCGGG GAGTTTAAACGCGGCCGCAGATCTAGCT-3′ [SEQID NO: 10] is ligated between fragment 3 and the HindIII site of pSP64(Promega Corporation, Madison, Wis.). Note that the BAV-1 sequences canbe cut out of this plasmid via the NotI restriction sites located in theflanking synthetic linker sequences.

Plasmid 1064-26

Plasmid 1064-26 contains a recombinant BAV-1 genome from which a portionof the E4 region (positions 33613 to 33725 from SEQ ID NO: 3) has beendeleted and replaced by a synthetic PstI site (5′-CTGCAG-3′) [SEQ ID NO:4]. The plasmid may be constructed according to the method above(Construction of Recombinant BAV-1 Genomes in E. coli). The homology DNAis derived from the NotI insert of plasmid 1054-93 and the adenoviralbackbone vector plasmid 990-50 is linearized by partial digestion withthe PvuI.

Plasmid 1066-29

Plasmid 1066-29 was constructed by inserting a BVDV g53 gene into theunique Pst1 site of plasmid 1055-93. The BVDV coding region was insertedin the reverse complimentary orientation such that it is transcribed bythe E4 region promoter located at the right end of the genome. The BVDVg53 gene was isolated according to the method above (Cloning of BovineViral Diarrhea virus g53 gene).

Plasmid 106644

Plasmid 1066-44 contains a recombinant BAV-1 genome from which a portionof to the E4 region (positions 29523 to 30403 from SEQ ID NO: 3) hasbeen deleted and replaced by a synthetic PstI site (5′-CTGCAG-3′) [SEQID NO: 4]. This plasmid may be used to generate recombinant bovineadenovirus vectors with gene insertions and/or a deletion at the E4region. The plasmid may be constructed according to the method above(Construction of Recombinant BAV-1 Genomes in E. coli). The homology DNAis derived from the NotI insert of plasmid 1055-93 and the adenoviralbackbone vector plasmid 990-50 is linearized by partial digestion withthe PvuI.

Plasmid 1066-51

Plasmid 1066-51 contains a recombinant BAV-1 genome from which theBAV-1's sequence from positions 30403 to 29523 [SEQ ID NO: 3] has beendeleted. The gene for the BVDV g53 (amino acids 1-394) was inserted intothe deleted region. The plasmid may be constructed according to themethod above (Construction of Recombinant BAV-1 Genomes in E. coli). Thehomology DNA is derived from the NotI insert of plasmid 1066-29 and theadenoviral backbone vector plasmid 990-50 is linearized by partialdigestion with the PvuI.

EXAMPLES Example 1 Sequence of BAV-1

By convention we will refer to the region containing the EcoR1 Afragment as the Left end of the BAV-1 genome. To complement thepreviously published restriction map (Maria Benko and B. Harrach, 1990Acta Veterinaria Hungarica 38:281-284) other restriction enzyme sites inthe BAV-1 genome were defined (PuvI, SmaI). The complete genome wascloned as several large restriction fragments. These included BamHI “B”,“C”, “D”, “F”, EcoR1 “A”, “E”, and PvuI “B”. The genome was also clonein its entirety as described above (Construction of Recombinant BAV-1Genomes in E. coli). These clones and 126 different oligonucleotideprimers were used according to the method described above (DNASequencing) to determine an over lapping sequence for the entire BAV-1genome.

This sequence (34,185 base pairs ) contains 43 methionine initiated openreading frames (ORF) of greater than or equal to 110 amino acids(excluding smaller nested ORFs). All 43 ORFs were compared to thecurrent version of the Genbank protein subset as described in themethods above (DNA Sequencing). Based on the BLAST analysis 28 of theORFs (ORFS 1-28) exhibited significant homology to one or more othervirus genes. Fifteen ORFs showed no significant homology to virus genesin the current version of Genbank (nORFs 1-15). Table 1 shows that theORFs have a widely varying homology to adenovirus genes from severaldifferent species.

TABLE 1 BAV-1 Left end Open reading frames (Orf) % ORF* Location** BestMatch to GenBank*** Similarity. ORF1 1400, 1867 BAV-2 E1A 49.5% ORF22189, 2656 BAV-2 E1B 58.6% ORF3 2566, 3777 SAV-3 E1B 42.3% ORF4 3838,4185 BAV-2 Hexon 36.9% ORF5 RC 4197, 5315 HuAd-7 Maturation Protein69.6% ORF6 RC 5285, 8530 BAV-2 Polymerase 72.9% ORF7 6255, 6680 HuAd-7unknown protein 59.4% ORF8 RC  8527, 10185 BAV-2 Terminal protein 71.2%ORF9 10376, 11437 CAV-1 Orf9 63.0% ORF10 11465, 13174 CAV-2 Hexonprotein 57.0% ORF11 13235, 14662 SAV-3 Penton base protein 74.6% ORF1214725, 15207 BAV-2 Major core protein 37.1% ORF13 15267, 16388 BAV-2Minor core protein 62.2% ORF14 16703, 17113 CAV-1 Minor capsid protein60.5% ORF15 17509, 20238 CAV-1 Hexon Late protein 2 75.3% ORF16 20241,20864 BAV-2 endoprotease 82.6% ORF17 RC 20906, 22246 OvAV DNA bindingprotein 77.8% ORF18 22258, 24498 BAV-3 Late 100 kd protein 59.0% ORF1924212, 24796 BAV-3 Late 33 kd protein 44.0% ORF20 25009, 25680 BAV-1Hexon protein 97.8% ORF21 25673, 26041 BAV-1 E3 12.5 kd protein 87.7%ORF22 25923, 27287 BAV-1 unknown protein 85.8% ORF23 27483, 29294HuAd-12 Fiber protein 31.2% ORF24 RC 29311, 29730 HuAd-40 E4 protein38.2% ORF25 RC 30404, 30739 HuAd-12 unknown protein 38.5% ORF26 RC30730, 31464 HuAd-40 E4 30 kd protein 28.9% ORF27 RC 31471, 32232 HuAd-9E4 34 kd protein 40.8% ORF28 RC 32956, 33384 AvAd dUTPase 54.7% nOrf1278, 736 nOrf2  697, 1167 nOrf3 5634, 5975 nOrf4 RC 10301, 10669 nOrf5RC 12607, 13212 nOrf6 RC 14246, 14722 nOrf7 RC 15479, 16102 nOrf8 RC17878, 18288 nOrf9 19031, 19621 NOrf10 21464, 21991 NOrf11 RC 24437,24820 nOrf12 RC 27800, 28174 nOrf13 RC 29523, 30407 nOrfl4 RC 32219,32557 nOrfl5 RC 33438, 33908 *RC, reverse compliment **positions on SEQID NO: 3 ***AvAd, Avian adenovirus; HuAD, Human Adenovirus; CAV, CanineAdenovirus, SAV, swine adenoviras; OvAd, sheep adenovirus

The E3 and E4 gene regions of BAV-1 can defined by homology to genesfrom the corresponding regions of the human adenoviruses. Evans et al(Virology 244:173-185) define the BAV-1 E3 gene region as bound by theTATA box sequence at positions 25362 to 25365 and the polyadenlyationsignal at positions 27291 to 27296. Analysis of the gene homologies fromTable 1 indicates that the E4 region of BAV-1 is bounded by thepolyadenylation signal at positions 29059 to 29065 and the TATA boxsequence at positions 34171 to 34174.

BAV-1 exhibits a complex sequence organization at its left and nightends. The genome exhibits an inverted terminal repeat (ITR) of 578 basepairs. A sequence of 419 base pairs is repeated twice at the left end ofthe genome. A single inverted copy of this repeat occurs at the rightend of the genome. The two 419 base pair repeats a the left end of thegenome are followed by a 424 bp sequence that appears as an invertedcopy upstream of the 419 base pair sequence at the right end of thegenome.

The sequence of the BAV-1 genome is useful for the construction ofrecombinant BAV-1 viral vectors For example this information may be usedby analogy to human adenovirus vector systems to predict non-essentialregions that may be used as gene insertion sites. The information mayalso be used to predict intergenic regions, which may also be used asgene insertion sites.

Example 2 Method of Constructing Recombinant BAV-1 Viral Vectors

We have developed a novel procedure for the generation of recombinantbovine adenovirus vectors. This procedure takes advantage of recombinantviral genomes constructed as bacterial plasmids (seemethods—Construction of Recombinant BAV-1 Genomes in E. coli. When DNAderived from these bacterial plasmids is transfected into theappropriate cells (see methods—Transfection of BAV-1 DNA) recombinantbovine adenovirus vectors are generated.

This procedure is exemplified by the infectivity of plasmid 990-50. DNAderived from this plasmid was transfected as described above into MDBKcells. Progeny viruses recovered from independent transfection stockswere amplified on MDBK cells and analyzed for growth characteristics,virus production yields, and DNA restriction patterns. In all cases,plasmid 990-50 derived adenovirus (S-BAV-002) was indistinguishable fromwild-type BAV-1.

This procedure can be used to generate bovine adenovirus vectorsexpressing useful foreign DNA sequences. The procedure may also be usedto delete genomic sequences from the bovine adenovirus vector. Theproduction of bovine adenovirus vectors bearing a bovine diarrhea virus(BVDV) glycoprotein E2 (g53) expression cassette and deletions in E4 andE3 regions of BAV-1 respectively are described below (see examples 4-6.

Example 3 Preparation of Recombinant Adenovirus Vector S-BAV-003

S-BAV-003 is a BAV-1 s that has a deletion in the E4 region of thegenome. This deletion spans the EcoR1 H and G fragments. This deletionremoves all or a major portion of ORFs 25-27 and nORF13. A poly linkersequence (GAATTCGAGCTCGCCCGGGCGAGCTCGAATTC) [SEQ ID NO: 15] containing aSmaI site was inserted into the deletion. As SmaI is absent from BAV-1genomic DNA (see FIG. 1), the introduction of this poly linker sequencecreates a useful unique SmaI site that may be exploited to directlyengineer the virus.

S-BAV-003 was created by transfection of DNA derived from plasmid1004-73.16.14 according to the method described above (Method ofconstructing recombinant BAV-1 viral vectors). The resulting viruseswere purified according to the method above (Plaque Purification ofRecombinant Constructs). Progeny viruses derived from independenttransfection stocks were amplified on MDBK cells and analyzed for BamH1,EcoR1, and SmaI DNA restriction patterns. This analysis indicates thatthe EcoR1 G and H fragments have been deleted and a SmaI site has beenintroduced into the genome. S-BAV-003 was also shown to grow to similartiters as the wild type BAV-1.

Example 4 Preparation of Recombinant Adenovirus Vector S-BAV-004

S-BAV-004 is a BAV-1 virus that has a deletion in the E4 region of thegenome. This deletion spans the EcoR1 H and G fragments. This deletionremoves all or a major portion of ORFs 25-27 and nORF13. The gene forthe bovine viral diarrhea virus (BVDV) glycoprotein 53 (g53) (aminoacids 1-394) under the control of the HCMV immediate early promoter wasinserted into the deleted region.

S-BAV-004 was created by transfection of DNA derived from plasmid1004-40 according to the method described above (Method of constructingrecombinant BAV-1 viral vectors). The resulting viruses were purifiedaccording to the method above (Plaque Purification ofRecombinant-Constructs).

Example 5 Preparation of Recombinant Adenovirus Vector S-BAV-005

S-BAV-005 is a BAV-1 virus that has a deletion in the E3 region of thegenome. The smaller SalI to BamH1 sub fragment of BamH1 fragment “B¹”(positions 25664 to 26850 from SEQ ID NO: 3) has been deleted. Thisdeletion removes a major portion of ORFs 21 and 22. A poly linkersequence (5′-TCGACAAGCTTCCC-3′) [SEQ ID NO: 16] containing a HindIIIsite was inserted into the deletion.

S-BAV-005 was created by transfection of DNA derived from plasmid1018-75 according to the method described above (Method of constructingrecombinant BAV-1 viral vectors). The resulting viruses were purifiedaccording to the method above (Plaque Purification of RecombinantConstructs).

Example 6 Preparation of Recombinant Adenovirus Vector S-BAV-006

S-BAV-006 is a BAV-1 virus that has a deletion in the E3 region of thegenome. The smaller SalI to BamH1 sub fragment of BamH-1 fragment B(positions 25664 to 26850 from SEQ ID NO: 3) has been deleted. Thisdeletion removes a major portion of ORFs 21 and 22. The gene for theBVDV g53 (amino acids 1-394) under the control of the HCMV immediateearly promoter was inserted into the deleted region.

S-BAV-006 was created by transfection of DNA derived from plasmid1018-42 according to the method described above (Method of constructingrecombinant BAV-1 viral vectors). The resulting viruses were purifiedaccording to the method above (Plaque Purification of RecombinantConstructs).

Example 7 Preparation of Recombinant Adenovirus Vector S-BAV-007

S-BAV-005 is a BAV-1 virus that has a deletion in the E3 region of thegenome. The smaller EcoR1 to BamH1 sub fragment of BamH1 fragment “B”(positions 25765 to 26850 from SEQ ID NO: 3) has been deleted. Thisdeletion removes a major portion of ORFs 21 and 22. A poly linkersequence (5′-TCGACAAGCTTCCC-3′) [SEQ ID NO:16] containing a HindIII sitewas inserted into the deletion.

S-BAV-007 was created by transfection of DNA derived from plasmid1018-45 according to the method described above (Method of constructingrecombinant BAV-1 viral vectors). The resulting viruses were purifiedaccording to the method above (Plaque Purification of RecombinantConstructs). Progeny viruses derived from independent transfectionstocks were amplified on MDBK cells and analyzed for BamH1, EcoR1, andXbaI DNA restriction patterns. S-BAV-007 was also shown to grow tosimilar titers as the wild type BAV-1.

Example 8 Preparation of Recombinant Adenovirus Vector S-BAV-014

S-BAV-014 is a BAV-1 virus that has a deletion in the E3 region of thegenome. The smaller EcoIR1 to BamH1 sub fragment of BamH1 fragment B(positions 25765 to 26850 from SEQ ID NO: 3) has been deleted. Thisdeletion removes a major portion of ORFs 21 and 22. The gene for theBVDV g53 (amino acids 1-394) under the control of the HCMV immediateearly promoter was inserted into the deleted region.

S-BAV-014 was created by transfection of DNA derived from plasmid1038-16 according to the method described above (Method of constructingrecombinant BAV-1 viral vectors). The resulting viruses were purifiedaccording to the method above (Plaque Purification of RecombinantConstructs). Expression of the BVDV g53 gene was assayed by the WesternBlotting Procedure. S-BAV-014 exhibited expression of a correct sizeprotein with specific reactivity to BVDV g53 antibody.

Example 9 Preparation of Recombinant Adenovirus Vector S-BAV-022

S-BAV-022 is a BAV-1 virus that has a deletion in the E4 region of thegenome. This deletion spans from positions 29523-30407 of SEQ ID NO: 3.A linker sequence 5′-CTGCAG-3′ containing a PstI site was inserted intothe deletion.

S-BAV-022 was created by transfection of DNA derived from plasmid1055-52 according to the method described above (Method of constructingrecombinant BAV-1 viral vectors). The resulting viruses were purifiedaccording to the method above (Plaque Purification of RecombinantConstructs). Progeny viruses derived from independent transfectionstocks were amplified on MDBK cells and analyzed for BamH1, EcoR1, andXbaI DNA restriction patterns S-BAV-022 was also shown to grow tosimilar titers as the wild type BAV-1.

Example 10 Preparation of Recombinant Adenovirus Vector S-BAV-023

S-BAV-023 is a BAV-1 virus that has a deletion in the E4 region of thegenome. This deletion spans from positions 29523-30407 of SEQ ID NO: 3.The gene for the BVDV g53 (amino acids 1-394) was inserted into thedeleted region. The BVDV g53 gene was under the control of E4promoter(s).

S-BAV-023 was created by transfection of DNA derived from plasmid1055-56 according to the method described above (Method of constructingrecombinant BAV-1 viral vectors). The resulting viruses were purifiedaccording to the method above (Plaque Purification of RecombinantConstructs). Expression of the BVDV g53 gene was assayed by the WesternBlotting Procedure. S-BAV-006 exhibited expression of a correct sizeprotein with specific reactivity to BVDV g53 antibody. Expression of theBDV g53 foreign antigen in S-BAV-023 establishes the utility of theBAV-1 E4 region promoter for transcription of foreign genes in vectorsystems.

Example 11 Preparation of Recombinant Adenovirus Vector S-BAV-025

S-BAV-025 is a BAV-1 virus that has a deletion in the E4 region of thegenome. This deletion spans from positions 33614-33725 of SEQ. ID NO: 3.A linker sequence 5′-CTGCAG-3′ containing a PstI site was inserted intothe deletion.

S-BAV-025 was created by transfection of DNA derived from plasmid1064-26 according to the method described above (Method of constructingrecombinant BAV-1 viral vectors). The resulting viruses were purifiedaccording to the method above (Plaque Purification of RecombinantConstructs). Progeny viruses derived from independent transfectionstocks were amplified on MDBK cells and analyzed for BamH1, EcoR1, andPstII DNA restriction patterns. S-BAV-025 was also shown to grow tosimilar titers as the wild type BAV-1.

Example 12 Preparation of Recombinant Adenovirus Vector S-BAV-026

S-BAV-026 is a BAV-1 virus that has a deletion in the E4 region of thegenome. This deletion spans from positions 29523-30403 of SEQ ID NO: 3.A linker sequence 5′-CTGCAG-3′ containing a PstI site was inserted intothe deletion.

S-BAV-026 was created by transfection of DNA derived from plasmid1066-44 according to the method described above (Method of constructingrecombinant BAV-1 viral vectors). The resulting viruses were purifiedaccording to the method above (Plaque Purification of RecombinantConstructs). Progeny viruses derived from independent transfectionstocks were amplified an MDBK cells and analyzed for BamH1, EcoR1, andXbaI DNA restriction patterns. S-BAV-026 was also shown to grow tosimilar titers as the wild type BAV-1.

Example 13 Preparation of Recombinant Adenovirus Vector S-BAV-027

S-BAV-027 is a BAV-1 virus that has a deletion in the E4 region of thegenome. This deletion spans from positions 29523-3043of SEQ ID NO: 3.The gene for the BVDV g53 (amino acids 1-394) was inserted into thedeleted region. The BVDV g53 gene was under the control of E4promoter(s).

S-BAV-027 was created by transfection of DNA derived from plasmid1066-51 according to the method described above (Method of constructingrecombinant BAV-1 viral vectors). The resulting viruses were purifiedaccording to the method above (Plaque Purification of RecombinantConstructs).

Example 14 Shipping Fever Vaccine

Shipping fever or bovine respiratory disease (BRD) complex is manifestedas a result of a combination of infectious diseases of cattle andadditional stress related factors (C. A. Hjerpe, The Bovine RespiratoryDisease Complex. In: Current Veterinary Therapy 2: Food Animal Practice.Ed. by J. L. Howard, Philadelphia, W.B. Saunders Co., 1986, pp670-680.). Respiratory virus infections, augmented by pathophysiologicaleffects of stress, alter the susceptibility of cattle to Pasteurellaorganisms that are normally present in the upper respiratory tract by anumber of mechanisms. Control of the viral infections that initiate BRDas well as control of the terminal bacterial pneumonia is essential topreventing the disease syndrome (F. Fenner, et at., “Mechanisms ofDisease Production: Acute Infections”, Veterinary Virology. AcademicPress, Inc., Orlando, Fla., 1987, pp 183-202.).

The major infectious diseases that contribute to BRD are: infectiousbovine rhinotracheitis virus, parainfluenza type 3 virus, bovine viraldiarrhea virus, bovine respiratory syncytial virus, and Pasteurellahaemolytica (F. Fenner, et al., “Mechanisms of Disease Production: AcuteInfections”, Veterinary Virology. Academic Press, Inc., Orlando, Fla.,1987, pp 183-202.) An extension of this approach is to combine vaccinesin a manner so as to control the array of disease pathogens with asingle immunization. To this end, mixing of the various BAV-1 vectoredantigens (IBR, BRSV, PI-3, BVDV and P. Haemolytica) in a single vaccinedose. Also, conventionally derived vaccines (killed virus, inactivatedbacterins and modified live viruses) could be included as part of theBRD vaccine formulation should such vaccine components prove to be moreeffective.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the fill scope ofequivalents to which such claims are entitled.

1. A recombinant virus comprising a foreign DNA sequence inserted into the E4 gene region of a bovine adenovirus.
 2. The recombinant virus of claim 1, wherein said foreign DNA encodes a polypeptide from a virus or bacteria selected from the group consisting of bovine rotavirus, bovine coronavirus, bovine herpes virus type 1, bovine respiratory syncytial virus, bovine para influenza virus type 3 (BPI-3), bovine diarrhea virus, bovine rhinotracheitis virus, bovine parainfluenza type 3 virus, Pasteurella haemolytica, Pasteurella multocida and/or Haemophilus somnus.
 3. The recombinant virus of claim 2, wherein said polypeptide comprises more than ten amino acids.
 4. The recombinant virus of claim 2, wherein said polypeptide is antigenic.
 5. The recombinant virus of claim 1, wherein said bovine adenovirus is a Subgroup 1 bovine adenovirus.
 6. The recombinant virus of claim 1, wherein said bovine adenovirus is a Subgroup 2 bovine adenovirus.
 7. The recombinant virus of claim 5, wherein said foreign DNA sequence is under control of a promoter located upstream of said foreign DNA sequence
 8. A mutant virus comprising a deletion of at least a portion of the E4 gene region of a bovine adenovirus.
 9. The mutant virus of claim 8 which also has a foreign DNA sequence inserted into the E4 gene region.
 10. The mutant virus of claim 8, wherein at least one open reading frame of said E4 gene region of said bovine adenovirus is completely deleted.
 11. A recombinant virus comprising a foreign DNA sequence inserted into the E3 gene region of a bovine adenovirus
 1. 12. The recombinant virus of claim 11, wherein said foreign DNA encodes a polypeptide from a virus or bacteria selected from the group consisting of bovine rotavirus, bovine coronavirus, bovine herpes virus type 1, bovine respiratory syncytial virus, bovine para influenza virus type 3 (BPI-3), bovine diarrhea virus, bovine rhinotracheitis virus, bovine parainfluenza type 3 virus, Pasteurella haemolytica, Pasteurella multocida and/or Haemophilus somnus.
 13. The recombinant virus of claim 12, wherein said polypeptide comprises more than ten amino acids.
 14. The recombinant virus of claim 12, wherein said polypeptide is antigenic.
 15. The recombinant virus of claim 12, wherein said foreign DNA sequence is under control of a promoter located upstream of said foreign DNA sequence.
 16. A mutant virus comprising a deletion of at least a portion of the E3 gene region of a bovine adenovirus
 1. 17. The mutant virus of claim 16 which also has a foreign DNA sequence inserted into the E3 gene region.
 18. The recombinant virus of claim 16, wherein at least one open reading frame of said E3 gene region of said bovine adenovirus 1 is completely deleted.
 19. A vaccine comprising the recombinant virus of claim
 1. 20. A method of inducing an immunological response in an animal, comprising introducing the vaccine of claim 19 to said animal.
 21. A vaccine comprising the recombinant virus of claim
 11. 22. A method of inducing an immunological response in an animal comprising introducing the vaccine of claim 21 to said animal. 